Compositions and methods for treatment of aortic fibrosis

ABSTRACT

The invention relates to compositions comprising vasoactive intestinal peptide (VIP) or fragments thereof, and the use of such compositions in the treatment of aortic fibrosis and other associated conditions.

TECHNICAL FIELD

This invention relates to compositions and methods for therapeutic orprophylactic treatment of aortic fibrosis. In particular this inventionconcerns compositions comprising VIP or certain active fragments of VIPand their use in the treatment of aortic fibrosis.

BACKGROUND

Any discussion of the prior art throughout the specification should inno way be considered as an admission that such prior art is widely knownor forms part of common general knowledge in the field.

The incidence of conditions such as stroke and dementia are more closelyrelated to the level of systolic blood pressure than diastolic bloodpressure. Elevations in systolic blood pressure have been thought tooccur as a consequence of decreased production of the intrinsicvasodilator endothelial derived relaxing factor (EDRF) or nitric oxide(NO) resulting in increased vascular tone or vasoconstriction. Thisexplains increases in systolic blood pressure but does not explain thefalling diastolic blood pressure and the widened pulse pressure that areassociated with increased cardiovascular risk. Only structural changesin the major blood vessel, the aorta, which reduce compliance and thusreduce the ability of the aorta to relax and absorb the systolicpressure wave as well as to undergo elastic recoil during cardiacfilling or diastole can explain this phenomenon. Loss of elastic fibresand their replacement by collagen as well as disruption of the smoothmuscle fibres by increased amounts of collagen and fibrous tissue resultin a decrease in elasticity and compliance. These changes in the aorticwall cause rigidity which in turn results in a reflectance wave inresponse to the pressure wave of cardiac systole. The effect of thereflectance wave is to augment and further increase systolic bloodpressure. The increased rigidity also prevents elastic recoil duringcardiac filling, attenuates the capacity of the vasculature to maintainblood pressure in diastole, and results in a lower diastolic pressure.The difference between systolic and diastolic pressures is termed thepulse pressure. A widened pulse pressure (>90 mmHg) denotes highabsolute risk for cardiovascular events such as stroke.

Currently available blood pressure lowering agents address the increasedvasoconstrictor component of systolic pressure and lower both systolicand diastolic pressures by similar amounts. The failure of these agentsto effect structural remodelling in the aorta means, that in patientswith a widened pulse pressure, treatment is limited by diastolicpressure reductions. As a consequence, systolic blood pressure oftenremains above recommended target levels. Thus pulse pressure remainswidened despite treatment or may be exacerbated by it and such patientsremain at high risk for cardiovascular events such as stroke.

There is thus a need for therapeutic agents which will prevent and/orreverse the structural changes to the aorta.

It is an object of the present invention to overcome or ameliorate atleast one of the disadvantages of the prior art, or to provide a usefulalternative.

SUMMARY OF THE INVENTION

The present invention is concerned with VIP and/or VIP fragments andtheir use in the treatment of aortic fibrosis. The compositions of thepresent invention have the ability to prevent the development, orreverse established fibrosis in the aorta and thus can be used fortherapeutic as well as prophylactic treatment.

According to a first aspect, the invention provides a composition forthe prophylactic or therapeutic treatment of aortic fibrosis, thecomposition comprising a pharmaceutically effective amount of vasoactiveintestinal peptide (VIP) (SEQ ID No. 1) or one or more functional VIPfragments selected from SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ IDNo. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, SEQ IDNo. 10, SEQ ID No. 11, SEQ ID No.12, SEQ ID No. 13, SEQ ID No. 14, SEQID No. 15, or SEQ ID No. 16.

Preferably, compositions according to the present invention areadministered in conjunction with a pharmaceutically acceptable carrier,which may be any of those known in the art or devised hereafter andsuitable for the intended use. As well as carriers, the pharmaceuticalcomposition of the invention may include other ingredients, includingdyes, preservatives, buffers and anti-oxidants, for example. They maypreferably be administered in conjunction with one or more other activeagents useful in the treatment of aortic fibrosis or heart conditions.They may, for preference, be formulated for administration by oral,intravenous, intramuscular or subcuticular routes.

Other methods of administration such as patches, snuffs, nasal spraysand the like, will be clear to those skilled in the art.

The pharmaceutically effective amount of VIP or an active VIP fragmentwill vary according to the patient and/or with the severity of thecondition to be treated. These variables can be ascertained by oneskilled in the art by routine experimentation. An appropriate dosagerange, as a starting point, can be derived from dosages administered inthe animal models described herein, or with reference toPCT/AU2005/000835 and PCT/AU2006/001869. The compositions of theinvention may be used to prevent or slow down progression of aorticfibrosis, as well as to reduce the degree of, or prevent establishmentof fibrosis.

According to a second aspect, the invention provides a method ofprophylactic or therapeutic treatment of aortic fibrosis in a subject,the method comprising administering to the subject at risk of developingaortic fibrosis, or to a subject having aortic fibrosis, a compositioncomprising a pharmaceutically effective amount of vasoactive intestinalpeptide (VIP) (SEQ ID No. 1) or one or more functional VIP fragmentsselected from SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5,SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9. SEQ ID No. 10,SEQ ID No. 11, SEQ ID No.12, SEQ ID No. 13, SEQ ID No. 14, SEQ ID No.15, or SEQ ID No. 16.

With respect to prophylactic treatment it will be understood that such atreatment would benefit subjects particularly who are at risk ofdeveloping aortic fibrosis. As an example of subjects in the riskcategory are those having associated conditions such as atherosclerosis,hypertension, chronic kidney disease, chronic vitamin D intoxication,vasculitis, diabetes, hypothyroidism, hyperlipidaemia, isolated systolichypertension, stroke, heart failure, myocardial infarction, end stagekidney failure, and aortic aneurysm.

Preferably, the prophylactic treatment is used to prevent or slow downthe development of fibrosis in a subject. The treatment may also be usedto prevent or slow down progression of established aortic fibrosis, oralternatively, to reduce the degree of established fibrosis.

Preferably, the method further comprises administration of one or moreother active agents useful in the treatment of aortic fibrosis.

It will be apparent to one skilled in the art that the pattern of use ofthe compositions of the invention may need to be altered for optimumeffect. It may be necessary to take into account the nature of thedisease or condition as well as its severity and any underlying risk orpredisposition factors.

Preferably VIP or one or more functional VIP fragments are administeredby a route selected from intravenous, intramuscular, by subcuticularinjection, oral, sublingual or nasal.

Preferably VIP or one or more functional VIP fragments are administeredin a dosage form selected from tablets, capsules, powders, liquidformulations, delayed or sustained release, patches, snuffs, nasalsprays and the like.

According to a third aspect, the invention provides vasoactiveintestinal peptide (VIP) (SEQ ID No. 1) or one or more functional VIPfragments selected from SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ IDNo. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, SEQ IDNo. 10, SEQ ID No. 11, SEQ ID No.12, SEQ ID No. 13, SEQ ID No. 14, SEQID No. 15, or SEQ ID No. 16, for use in the prophylactic or therapeutictreatment of aortic fibrosis.

Preferably, the use is to prevent or slow down progression ofestablished aortic fibrosis. Alternatively, the use is to prevent orslow down the development of fibrosis in a subject or an at risk group.Also preferably, the use is to reduce the degree of establishedfibrosis.

Preferably, the use further comprises administration of one or moreother active agents useful in the treatment of aortic fibrosis.

According to a fourth aspect, the invention provides use of vasoactiveintestinal peptide (VIP) (SEQ ID No. 1) or one or more functional VIPfragments selected from SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ IDNo. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8. SEQ ID No. 9, SEQ IDNo. 10, SEQ ID No. 11, SEQ ID No.12, SEQ ID No. 13, SEQ ID No. 14, SEQID No. 15, SEQ ID No. 16 in the manufacture of a medicament for theprophylactic or therapeutic treatment of aortic fibrosis.

The term “prophylactic” as used in the context of the present inventionis intended inter ilia to encompass treatments used to prevent or slowdown the development of aortic fibrosis in the at risk group. Highproportion of subjects that may be given prophylactic treatment mayalready have signs of aortic fibrosis or heart disease.

According to a fifth aspect, the invention provides a method of reducingcollagen formation or enhancing collagen degradation in the aorta of asubject, the method comprising administering to the subject acomposition comprising a pharmaceutically effective amount of vasoactiveintestine peptide (VIP) (SEQ ID No. 1) or one or more functional VIPfragments selected from SEQ ID No. 2, SEQ ID No. 3, SEQ ID No. 4, SEQ IDNo. 5, SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8, SEQ ID No. 9, SEQ IDNo. 10. SEQ ID No. 11, SEQ ID No.12, SEQ ID No. 13, SEQ ID No. 14, SEQID No. 15, or SEQ ID No. 16.

Unless the context clearly requires otherwise, throughout thedescription and the claims, the words “comprise”, “comprising”, and thelike are to be construed in an inclusive sense as opposed to anexclusive or exhaustive sense; that is to say, in the sense of“including, but not limited to”.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Aortic fibrosis in rats after 4 weeks infusion of vehiclecontrol or peptide at 5 pmol/kg/min.

FIG. 2: Shows the effect of 4 weeks treatment with VIP and various VIPfragments at 5 pmol/kg/min in the SHR on fibrosis within the aorticwall. * p<0.005, ** p<0.0005 vs Control infusion.

FIG. 3: Shows the effect of 4 weeks treatment with various VIP fragmentsat 5 pmol/kg/min in the SHR on fibrosis within the aortic wall. *p<0.025, ** p<0.01, *** p<0.005 and **** p<0.001 vs Control infusion.

FIG. 4: Shows the effect of 4 weeks treatment with various VIP fragmentsat 5 pmol/kg/min in the SHR on fibrosis within the aortic wall.

DESCRIPTION OF THE PREFERRED EMBODIMENT

It has now been found that the VIP molecule as a whole, acts to prevent,reduce or reverse aortic fibrosis. Further, in view of the well acceptedviews held in this field, it has surprisingly been found that VIPfragments lacking amino acids and motifs thought to be important fortheir function are nevertheless useful therapeutic agents that reverseor delay the onset of aortic fibrosis, or prevent onset of fibrosis insubjects at risk of developing heart disease. Particularly useful VIPfragments can be selected from, but not limited to, VIP(4-12),VIP(4-16), VIP (4-10), VIP (4-20). VIP (4-24), VIP (10-28), VIP (16-20),VIP (16-24), VIP (16-28). VIP (6-10), VIP (6-12), VIP (6-16), VIP(6-20), VIP (6-24), and VIP (6-28).

The use of the pharmaceutical compositions of the invention in thetreatment of aortic fibrosis represents a new class of therapeuticagents for these conditions. Currently there are no existing treatmentsfor aortic fibrosis. Without wishing to be bound by any particularmechanism of action, it is believed that the pharmaceutical preparationsof the invention may target virtually all the currently known promotersof aortic fibrosis.

On the basis of the present studies, and not wishing to be bound bytheory, it is postulated that VIP or VIP fragments act as majorregulators to prevent the development of fibrosis, and that thedepletion of VIP may unleash the synthesis of a number of profibroticmediators, thereby causing aortic injury. The VIP fragments of thepresent invention seem to be able to act in much the same way as thenative VIP but are more suited for therapeutic applications due tosmaller size and hence increased stability and ease of manufacture.

It will be understood that the present invention also encompasses withinits scope certain analogues of the VIP fragments, which are based onconservative substitutions of one or more amino acids of the VIPfragments, with amino acids which do not alter the biological activitiesof the VIP fragments. Such substitutions would be well known to thoseskilled in the art and would not require more than simpletrial-and-error using well-established techniques. Hence, the term “VIPfragment” as used in the context of the present invention is intended toencompass such analogues.

All the sequences relate to VIP and fragments of human origin, but dueto the very high level of amino acid conservation, VIP and fragmentsthereof derived from other mammalian species are also contemplated andencompassed by the present invention.

The present invention also contemplates pharmaceutical compositions,which include VIP and/or active VIP fragments. Such compositions mayinclude any type of dosage form such as tablets, capsules, powders,liquid formulations, delayed or sustained release, patches, snuffs,nasal sprays and the like. The formulations may additionally includeother ingredients such as dyes, preservatives, buffers andanti-oxidants, for example. The physical form and content of thepharmaceutical formulations contemplated are conventional preparationsthat can be formulated by those skilled in the pharmaceuticalformulation field and are based on well established principles andcompositions described in, for example, Remington: The Science andPractice of Pharmacy, 19^(th) Edition, 1995; British Pharmacopoeia 2000,and similar formulation texts and manuals. The compositions of thepresent invention may also include other active agents useful in thetreatment of aortic fibrosis.

The route and frequency of administration of the compositions of thepresent invention will depend on the treatment requirements and thenature of the molecule to be administered. Thus the formulations may besuitably prepared for administration by intravenous, intramuscular orsubcuticular injection. VIP and/or VIP fragments may also be suitablefor mucosal administration such as oral, sublingual, nasal and the like.These parameters are easily established by those skilled in the art.

The pharmaceutical compositions of the invention have been shown to beeffective in preventing or slowing down progression of aortic fibrosis,as well as in reducing the degree (reversal) of established fibrosis andthus are important in therapeutic applications. The compositions of thepresent invention are therefore useful for prophylactic or therapeutictreatment of aortic fibrosis. These are important findings with respectto the range and severity of conditions, which can be treated with thecompositions of the present invention.

The compositions of the present invention may be used prophylacticallyin subjects at risk of developing aortic fibrosis. As an example ofsubjects in the risk category are those having associated conditionssuch as atherosclerosis, hypertension, chronic kidney disease, chronicvitamin D intoxication, vasculitis, diabetes, hypothyroidism,hyperlipidaemia, isolated systolic hypertension, stroke, heart failure,myocardial infarction, end stage kidney failure, aortic aneurysm and thelike.

By conserving the VIP content of the aorta in a subject with, or at riskof developing aortic fibrosis, through the use of the compositions ofthe present invention, significant therapeutic benefits can be achievedincluding reduction of fibrosis, reduction in the level, production oractivity of pro-fibrotic mediators, reduction in progression offibrosis, reduction in collagen formation or enhancing collagendegradation in the aorta.

The invention will now be described more particularly with reference tonon-limiting examples.

EXPERIMENTAL

All general methodology and techniques have been described in detail inPCT/AU2005/000835, incorporated in its entirety herein by reference.

Example 1 Amino Acid Sequence of VIP and VIP Fragments

SEQ ID No 1: VIP(1-28)-His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr- Leu-Asn-Ser-Ile-Leu-AsnSEQ ID No 2: VIP(4-12)-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg SEQ ID No 3:VIP (4-16)-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg- Leu-Arg-Lys-GlnSEQ ID No 4: VIP (4-10)-Ala-Val-Phe-Thr-Asp-Asn-Tyr SEQ ID No 5:VIP (4-20)-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Met-Ala-Val-Lys SEQ ID No 6:VIP (4-24)-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Asn SEQ ID No 7:VIP (10-28)-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile-Leu-Asn SEQ ID No 8:VIP (16-20)-Gln-Met-Ala-Val-Lys SEQ ID No 9:VIP (16-24)-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Asn SEQ ID No 10:VIP (16-28)-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Asn- Ser-Ile-Leu-AsnSEQ ID No 11: VIP (6-10)-Phe-Thr-Asp-Asn-Tyr SEQ ID No 12:VIP (6-12)-Phe-Thr-Asp-Asn-Tyr-Thr-Arg SEQ ID No 13:VIP (6-16)-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg- Lys-Gln SEQ ID No 14:VIP (6-20)-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg- Lys-GIn-Met-Ala-Val-LysSEQ ID No 15: VIP (6-24)-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Asn SEQ ID No 16:VIP (6-28)-Phe-Thr-Asp-Asn-Tyr-Thr-Arg-Leu-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Ile- Leu-Asn

Example 2 Effect of VIP Fragment Infusion on Aortic Fibrosis in RatModels of Fibrosis

Two animal models of aortic fibrosis were used (animals obtained fromAustralian Animal Resources, Perth, Western Australia, Australia)

-   i) Male SHR rats on 2.2% salt diet-   ii) Male WKY rats on 4.4% salt diets-   In each model the rats were randomised to VIP(1-28), VIP(4-12),    VIP(4-16), VIP (4-10), VIP (4-20), VIP (4-24), VIP (10-28), VIP    (16-20), VIP (16-24), VIP (16-28), VIP (6-10), VIP (6-12), VIP    (6-16), VIP (6-20), VIP (6-24), and VIP (6-28). All peptides were    obtained from or synthesised by Auspep, Australia. VIP fragments    were dissolved in Hartman's solution for subsequent infusion    studies.-   Commencing at 12 weeks of age, the rats were acclimatized to tail    cuff blood pressure measurements and handling for 2 weeks. They then    underwent operative insertion of an osmotic minipump (Alzet) which    was designed to deliver vehicle alone (Hartman's solution, (Baxter    Health Care Corporation)—(Controls)) or VIP, VIP fragment at a dose    of 5 pmol/kg/min intravenously.

The infusion was continued for 4 weeks, during which the rats wereweighed and their blood pressures measured twice weekly. At the end ofthe 4 week infusion period, the rats were anaesthetized and their aortasharvested.

After fixation in buffered formalin, the aortas were embedded in wax,sectioned and stained with haematoxylin and eosin or with MassonTrichrome (Lomb Scientific).

For quantitation of aortic fibrosis, twenty fields from each aorta weredigitized and the amount of fibrosis in each determined as percentsurface area using Image-Pro Plus V5.0 software. The mean value for eachrat and subsequently for each infusion group was then determined.

FIGS. 1 to 4 show reductions in aortic fibrosis which occurred as aresult of the infusion for 4 weeks of VIP and various VIP fragments inthe SHR rats on a 2.2% salt diet.

The importance of the present invention to health care will beimmediately apparent to one skilled in the art upon reading thisdisclosure. The pharmaceutical preparations of the invention, which actto prevent the progression of the underlying lesion (fibrosis), or evenreverse fibrosis, have the capacity to prevent the escalation of mild tosevere disease and hence to substantially reduce the health care burden.The overall size of certain VIP fragments and their activity makes themideally suitable as targets for drug development.

It is to be appreciated that other embodiments and variants of thecompositions, methods and uses of the invention, in keeping with theteaching and the spirit of the invention described, are contemplated andthat these are within the scope of the invention.

What is claimed is:
 1. A method of prophylactic or therapeutic treatmentof aortic fibrosis in a subject, the method comprising administering tothe subject at risk of developing aortic fibrosis, or to a subjecthaving aortic fibrosis, a composition comprising a pharmaceuticallyeffective amount of vasoactive intestinal peptide (VIP) (SEQ ID NO:1) orone or more functional VIP fragments selected from SEQ ID NO:2, SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8,SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQID NO:14, SEQ ID NO:15, or SEQ ID NO:16.
 2. The method according toclaim 1, wherein the treatment prevents or slows down the development offibrosis in a subject.
 3. The method according to claim 2, wherein thetreatment prevents or slows down progression of established aorticfibrosis, or reduces the degree of established fibrosis.
 4. The methodaccording to claim 1, further comprising administration of one or moreother active agents useful in the treatment of aortic fibrosis.
 5. Themethod according to claim 1, wherein VIP or one or more functional VIPfragments are administered by a route selected from intravenous,intramuscular, by subcuticular injection, oral, sublingual or nasal. 6.The method according to claim 1, wherein VIP or one or more functionalVIP fragments are administered in a dosage form selected from tablets,capsules, powders, liquid formulations, delayed or sustained release,patches, snuffs, nasal sprays and the like.
 7. A method of reducingcollagen formation or enhancing collagen degradation in the aorta of asubject, the method comprising administering to the subject acomposition comprising a pharmaceutically effective amount of vasoactiveintestine peptide (VIP) (SEQ ID NO:1) or one or more functional VIPfragments selected from SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10,SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, orSEQ ID NO:16.
 8. A composition for the prophylactic or therapeutictreatment of aortic fibrosis, the composition comprising apharmaceutically effective amount of vasoactive intestinal peptide (VIP)(SEQ ID NO:1) or one or more functional VIP fragments selected from SEQID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, or SEQ ID NO:16.